1. Technical Field of the Invention
The present invention relates, generally, a method for controlling pathogens in lawn and garden soils using an induced formation of chitinase.
More particularly, the present invention provides a method for inducing formation of chitinase enzyme in soil to degrade the chitin present in fungi and insects, e.g., nematodes, and to minimize their destructive effect on lawn and garden vegetation.
Chitinase enzymes can be readily produced by aerobic fermentation in the presence of soil microbes, which produce enzymes and biodegradation products, which, in turn, can be used as a bio-pesticide/fertilizer.
2. Description of the Prior Art
Soil contains a diversity of life forms which can interact with plants, such life forms including bacteria, fungi and nematodes. These biological forms are particularly abundant in the rhizosphere, the area of soil that surrounds and is influenced by the plant roots. Rhizobacteria are those bacteria which are adapted to the rhizosphere. There is a complex interaction among the various life forms in the soil, where some are antagonistic and others are mutually beneficial. Similarly complex is the interaction between the plants and the soil life forms, which can helpful to the plant in some instances, and harmful in others. Nematode pathogens harmful to plants include species from the genera Meloidogyne, Heterodera, Ditylenchus, and Pratylenchus. Nematode-caused plant diseases include root galls, root rot, lesions, “stubby root,” stunting, as well as other rots and wilts resulting from pathogenic fungi effects on nematode-weakened plants. Fungal species that are harmful to plants come from a wide variety of genera, including Fusarium, Pythium, Phytophthora, Verticillium, Rhizoctonia, Macrophomina, Thielaviopsis and Scierotinas. Plant diseases caused by fungi include pre- and post-emergence seedling damping-off, hypocotyl rots, root rots, crown rots and vascular wilts.
The exoskeleton structure of insects is characterized by the presence of chitin, which is also an essential element in the hyphae of fungi, including the Ascomycetes, Basidiomycetes and Zygomycetes. Chitin is a nitrogen-containing biopolymer, a polysaccharide, consisting of unbranched chains of -(1,4)-acetamide-2-deoxy-D-glucose, also termed poly(N-acetyl-D-glucosamine.) Chitin is found in fungi and arthropods, where it is a principal component in the exoskeletons. In nematodes it not only covers the adult form, but the eggs and cysts as well. Chitin may be regarded as a derivative of cellulose, the main structural support of trees and plants, in which the C-2 hydroxyl groups have been replaced by acetamide residues. The term chitinase refers to the various forms of the family of hydrolytic enzymes that catalyze the depolymerization of chitin by endolytic and exolytic mechanisms. The endochitinases, represented by many chitinases in plants, cleave the internal -1,4-glycosidic linkages of chitin to produce oligomers of at least three monomeric units. The exo-chitinases, such as the bacterial chitinases elaborated by Serratia marcescens, hydrolyze terminal monomer linkages. In addition, chitin can be deacetylated to chitosan with chitin deacetylase. That leaves a polymer chain with an amino group on each glucose linkage.
The major source of chitin, however, is marine fauna, generally as a by-product of the seafood industry. These include crab, lobster and shrimp shells. As a raw material, chitin is, therefore, plentifully available and practically free. Huge amounts of chitin are thrown away each year as a waste material. Purified chitin is relatively expensive to obtain, however, since seafood shells are tough composite materials, and by their very nature resistant to chemical attack. Thus, a vigorous extraction procedure must be applied to obtain the pure material.
Chitin is synthesized extensively by insects at the end of their larval stage and at transitions in their various life cycles, such as wing and new exoskeleton formation after shedding. Because chitinase degrades chitin, it thereby acts as a biocide for insect and fungal pathogens. The degradation of chitin makes insects more vulnerable to various toxicants, microorganisms and parasites which can penetrate the chitin-reduced exoskeleton. Environmental chitinase can also interfere with life cycle changes in insects, such as pupae formation.
To take advantage of this degradative activity, natural evolutionary processes have led to the development of genetic structures in certain plants, plant seeds and fungi which code for the production of chitinase. For example, chitinase has been shown to be induced in rhizoctonia solani-infected rice plants. Also, a number of fungi which are pathogenic to insects have been demonstrated to produce and use chitinase as part of their invasion of their insect hosts.
Harman et al., U.S. Pat. No. 5,173,419, has isolated and characterized the chitinases from trichoderma harzianum which inhibit the activity of chitin-containing fungi and insects. These chitinases can be applied to plants, or to the surrounding soil, in order to protect them. Harman et al. suggests that the genes coding for the chitinases can be isolated and, for example, inserted into the genome of a microorganism to provide a transgenic microorganism capable of producing chitinase as a biocontrol agent.
In bacteria, an induced chitinase enzyme level has been demonstrated in a specific strain of Serratia, where higher chitinase yields were produced by the strain when chitin was present in the medium. Suslow et al. have disclosed novel bacterial strains and/or plants which are created by the introduction of foreign DNA linked to a sequence encoding for the production of chitinase, for the purpose of inhibiting plant pathogens. See, U.S. Pat. Nos. 4,751,081; 4,940,840; 5,290,687; 5,374,540; 5,554,521; 5,633,450; and, 5,776,448.
For example, genetically-modified wheat has demonstrated the ability to prevent fungal infection by reducing the attachment of spores and their ability to invade. As a specific example of bacterial biocontrol of a fungal patho-pathogen, it has been demonstrated in the prior art (Kobayashi and El-Barrad 1996) that a newly-classified bacterium, Lytobacter kobii, is capable of controlling summer patch disease of Kentucky bluegrass through the production of a number of extracellular lytic enzymes, including chitinase and -1-3-glucanase. These directly damage fungal tissue and are believed to contribute to the organism's ability to parasitize the turf pathogen (M. Poae.)
It has also been reported in the art that pregnant thoroughbred mares in Kentucky have been losing hundreds of their foals as a result of an illness that scientists believe to be a result of a fungus growing on the State's famous bluegrass. The patch-disease fungus is known to produce a number of toxins which are lethal to grazing animals, such as horses and cattle.
The adaptation of soil bacteria to produce chitinase from chitin can be inferred from a study in 1982, conducted Auburn University's Agricultural Experiment Station, which showed that the addition of chitin to soil had an inhibitory, or direct, biocidal effect on soil nematodes. Biodegradation of the chitin is believed to result in high soil ammonia, which is toxic to many nematode species. But the soil bacteria also elaborated chitinase, which attacks the protective, chitinous outer layer of nematode eggs. That same study also revealed that chitin added to soil at loading rates of 2% inhibited seed germination in a number of plants, which effect has been ascribed to the inhibition of necessary fungal degradation of the outer seed coating. It is also possible that excess ammonia or nitrate levels produced by the degradation of nitrogenous chitin was causing phytotoxicity.
An attempt to harness the induced production of chitinase by a growing plant was provided recently by Stoner et al., U.S. Pat. No. 6,193,988, which disclosed a disease-controlling planting system comprising a plurality of parts. A typical system disclosed by Stoner et al. would include a propagule (e.g. a potato eye), chitin and chitosan (deacylated chitin)—to trigger the release of a naturally defensive substance from the propagule to protect itself from disease, a non-gaseous communication medium, and an encapsulant, in which the propagule can develop until it is able to withstand disease on its own. The natural defensive systems said to be released would include chitinase, beta-1,3-glucanase, protease inhibitors, phenylalanine lyase, chitosanase, and PR1-5 proteins.
Homeowners and other people concerned with maintaining home gardens and lawns, as well as turf such as golf courses to which the general public can be exposed, are continuously and increasingly concerned about the use of chemical pesticides and fertilizers for their proper control. And, for produce grown in these gardens, consumers have expressed a desire for organically-based pest control agents. In addition, the use of chemical pesticides, although often fairly effective, has been accompanied by other problems. The broadcast application on a field basis may not provide the uniform and concentrated amounts necessary at the particular plant. Air currents may carry these chemicals to neighboring areas, to the potential detriment of local inhabitants. And, to the extent the pesticide does not break down and remains in the soil, it may produce by-products, or residual pesticide which can pose a problem of contamination. As an example of the concern regarding the use of chemical pesticides in the home environment, the Legislature of New York State recently enacted a law mandating that the application of registered pesticidal and herbicidal chemicals to home lawns, by companies that supply such services, must be preceded by a pre-notification to neighboring homeowners, in order to allow them the opportunity to avoid such contact by family members and pets. Thus even registered products, which must meet some level of acceptable safety, are recognized as having inherent toxicological concern. No such warnings are required when such materials are applied to agricultural soils, where population densities are lower, and where the involved parties are generally more attuned to attendant risks of synthetic chemicals.
Most methods currently known to the prior art for producing chitinase enzymes rely upon procedures for genetically modifying plants via the introduction of various genes into plants, microbes and/or a fermentation process based upon use of chitin in its pure form, since various forms of microbes will typically regulate gene expression for adapting to the environment and, thereby, cease chitinase production when other, often more amenable, sources of nitrogen and carbon are available in the environment.
Public resistance to the use of genetically-modified organisms (“GMOs”) is increasing, particularly (and not surprisingly!) in countries within the European Union. As a consequence, the public debate regarding the commercialization of GMOs may limit practical marketability and otherwise necessary usage. In developing countries, the costs associated with the need for purified chitin for such fermentation poses a significant problem, in terms of economic viability, of a technology which competes with conventional fertilizer and pesticides. Current pricing of purified chitin can be as high, if not higher, than $(US)20/lb.
The use of raw shrimp shells poses a problem, as well, for the production of chitinase, since their primary use is in the food industry, where fresh shrimp shells contain too high a percentage of water to compete with the commercial chemicals used for fertilizer and pesticide.
The only viable economic source—dried shrimp shell—have two drawbacks which limit their use for such applications:
(1) Dried shrimp shells produce a pungent and unpleasant odor, which makes the direct use of such material, especially for domestic lawn and garden care, impractical from a commercial standpoint.
(2) Dried shrimp shells contain up to 40% protein, some of which are known allergens; a powerful argument against their use for applications outside of fish food for pisciculture. Additionally, proteins delay the onset of chitinase enzyme production by the fermentation microbial community, since the proteins are a more desirable carbon and nitrogen source.
The present invention is, therefore, the result of a search for a natural means for maintaining, controlling, protecting and feeding lawns, turf and home gardens, and thereby replacing chemical pesticides, which are facing increasing disfavor and concern among the general populace. More particularly, the presently claimed invention is concerned with the efficacy of naturally-generated chitinases as biocontrol agents, and whether it is possible to control this powerful natural system as a substitute for the more troublesome chemical pesticides in reducing pathogens in plant growth areas of high contact with humans. The term “turf” as will be used in the present Specification should be understood to include the broad expanses of grasses, such as in golf courses and public parks, where exposure to large numbers of persons is common.
This need, which is met by the present invention, has produced chitinase-related compositions and their methods for use, which unexpectedly combine the desired attributes of natural biocidal activity, herbicidal action, and soil fertilization with low toxicity and allergenicity. The invention derives from the adaptive tendency of certain soil bacteria to generate a class of gycolytic enzymes, particularly chitinases, from flaked or particulated chitin, a naturally-occurring by-product of the seafood industry. It also includes, the fortuitous creation of nitrogenous fertilizing materials resulting from the degradative action of the induced chitinase on the chitin feedstock. These and other aspects of the present invention will become evident in this Specification.